Toxoplasma gondii is a widespread apicomplexan parasite that causes life-threatening disease in neonates and immunocompromised individuals. T. gondii and other apicomplexan parasites use a unique form of gliding motility to invade into and escape from cells of their hosts and to disseminate throughout the body during infection. It is well established that T. gondii MyosinA (TgMyoA) plays an important role in parasite motility, and parasites lacking TgMyoA are completely avirulent. How TgMyoA and the proteins with which it interacts work together to drive motility is therefore a fundamentally important question. Recent data have called into question the current ?linear motor? model of motility and suggested the existence of a parallel, non- TgMyoA-based motility mechanism. This project will fill in a key gap in our understanding of parasite motility by determining the directionality of the forces generated by a parasite as it moves through the extracellular matrix. Capitalizing on our experience in high-speed imaging and analysis of parasite 3D motility and on our longstanding collaboration with mathematician Mark Rould, the Specific Aims of the project are to: (1) Develop 3D matrix deformation mapping methods to visualize the directionality of the forces moving parasites exert on the surrounding extracellular matrix and (2) Use 3D matrix deformation mapping to test key predictions of the linear motor model of T. gondii motility and to determine how disruption of the motor machinery affects the ability of the parasite to generate force. Motility plays a central role in the life cycle and virulence of T. gondii, yet the underlying mechanisms remain controversial. Understanding how the parasite generates force and determining whether it can use alternative mechanisms in the absence of TgMyoA will be critical to developing new, effective drugs that target parasite motility. The innovative methods developed here will be used to test key tenets of the current model of parasite motility, bring clarity to the role of TgMyoA and enable new approaches to analyzing motility based on the parasite's ability to generate force. Since the proteins and mechanisms underlying motility appear to be conserved among apicomplexans, the new mechanistic insights generated by this study and the future work it enables are likely to be directly relevant not just to T. gondii, but to other apicomplexan parasites as well. .